To understand how a pathogenic mutation causes inherited eye disease, it is necessary to recognize how pathogenic mutations could affect protein structure-function, metabolic pathways, and how these perturbations could be associated clinical parameters describing the disease phenotype. For this purpose, we express and purifying proteins targeted by missense mutations in inherited eye disease with a purpose to mimic the effect of pathogenic mutations in vitro. Also, we are trying to develop computational methods, perform molecular modeling of atomic protein structures, and predict the effect of missense changes from the inherited disease. Results of this analysis are presented at the ocular proteome website. This year we successfully purified and analyzed the function of the full-length and truncated versions of human recombinant tyrosinase-related protein 1, TYRP1 and TYRP1tr. We also developed an effective method for the prediction of missense mutation severity in multidomain proteins from inherited disease. Tyrosinases are melanocyte specific enzymes involved in melanin biosynthesis. Mutations in their genes cause oculocutaneous albinism associated with reduced or altered pigmentation of skin, hair, and eyes. The recombinant human intra-melanosomal domains of tyrosinase, TYRtr(19-469), and TYRP1tr (25-472), and their full-length variants were studied in vitro to define their functional relationship (Dolinska et al, PCMR, 2019). Proteins were expressed or coexpressed in whole Trichoplusia ni larvae and purified. Their associations were studied using gel filtration and sedimentation equilibrium methods. Protection of TYRtr was demonstrated for the first time by measuring the kinetics of tyrosinase diphenol oxidase activity in the presence (1:1 and 1:20 molar ratios) or the absence of TYRP1tr for 10 hr under conditions mimicking melanosomal and ER pH values. Our data indicate that TYRtr incubation with excess TYRP1tr protects TYR, increasing its stability over time. However, this mechanism does not appear to involve the formation of stable heterooligomeric complexes to maintain the protective function of a protein. Our finding could be important for understanding the functions of tyrosinases in melanogenesis. To determine the specific molecular mechanism underlying tyrosinase interactions, we analyzed the potential interaction between the proteins using several different model systems in both in vitro and in vivo conditions. Using gel-filtration and sedimentation equilibrium methods, we illustrated that TYRP1tr is a monomeric glycoprotein with a weight-average molecular weight of 60 kDa and does not form stable homo-oligomers. Previous studies in vivo suggested that TYRP1 functions as a molecular chaperone for TYR in the ER, which suggests that interactions of TYRP1 with TYR occur in the ER and that homo-oligomerization of TYR is a step-in proper protein maturation within the ER. However, this idea could be easily criticized. First, there is no direct observation that TYRP1 helps tyrosinase maintain a native protein fold in vitro. However, the lectin chaperones calnexin and calreticulin, which are components of the ER quality control system associate with TYR to help the protein folding. Second, there is no observation of TYRP1 consuming energy to perform the protective function, in contrast to chaperone-like molecules requiring ATP or GTP as an energy source to perform their function. Third, according to our in vitro data, TYRP1 is monomeric and does not form a large oligomeric homo-complex to provide a protein surface for the binding of misfolded proteins, as suggested previously. From this view, the mechanism of the TYRP1 protection could be different from that of classical chaperone function. In addition, we observed an improvement in catalytic activity and a threefold increase in TYRtr efficiency with a 20fold excess of TYRP1tr at the pH of melanosomes (pH 5.5). The significant excess of TYRP1tr led us to the possible explanation that its protective effect could be related to molecular crowding (Minton, 2001, 2006), which is a mechanism that has been frequently reported for other proteins. Molecular crowding has been shown to enhance the protein stability, intrinsic catalytic efficiency, and structure of proteins. Crowding is characterized by molecular volume exclusion due to the excess of a highly concentrated protecting protein with decreases the available space ligands may occupy, bringing them in closer proximity to a corresponding active site, and complex pattern of interatomic interactions at protein surfaces of apoferritin (Sergeev et al., 2018). Therefore, molecular crowding might provide an explanation for the improvement in TYRtr function with an excess of protecting protein, TYRP1tr. One of the challenging tasks in the analysis of genetic alterations is related to the absence structure of multi-domain proteins, some of which contained up to several hundred structural domains and atomic structure are not available. These proteins account for 70% of the eukaryotic proteome and in the majority are very difficult for computational analysis. In genetic disease, multi-domain proteins are affected by numerous missense mutations. The mutation effects on protein stability and their roles in genetic disease are unspoken. With a purpose to understand how to analyze such proteins, we selected proteins targeted by genetic alterations in inherited eye disease (Wood Ortiz & Sergeev, Scientific Reports,2019). The domain stability evaluation was performed for nine proteins, Eyes Shut homolog, Fibrillin-1, Fibrillin-2, Complement Factor H, Protocadherin-15, Protocadherin Fat 1, Protocadherin Fat 4, Roundabout homolog 3, and Cadherin-23. Alterations in these proteins cause genetic eye diseases such as retinitis pigmentosa, age-related macular degeneration, and others. To simplify the analysis, the proteins were split into individual domains. Each domain structure was built using homology modeling and then divided into 7 groups using protein fold similarities. These domain groups were epidermal growth factor-like, laminin globular, sushi, immunoglobulin-like C2-type, fibronectin type-III, cadherin, and transforming growth factor-beta. In total, the 291 protein domain structures were individually homology-modeled, equilibrated using 2 ns molecular dynamics in water to achieve better domain stereochemistry, and subjected to global computational mutagenesis to evaluate the effect of mutations on the protein stability. Mutation propensities within each group of domains were then averaged to find residues critical for protein stability of domain fold. The consensus derived from the sequence alignment shows that the critical residues determined by global mutagenesis are conserved within each group. From the global mutagenesis, we concluded that 80% of known disease-related genetic variants are associated with the residues critical for proper maintaining of protein fold and are expected to have significant destabilizing effects on domain structure. Our work provides an in-silico quantification of protein stability and could help to analyze the complex relationship among missense mutations, multi-domain protein stability, and disease phenotypes in inherited eye disease. Results of analysis of multi-domain proteins were incorporated in the ocular proteome web-site at the NEI Commons (https://neicommons.nei.nih.gov/#/proteomeData). The latest version of the ocular proteome web site contains in-silico predictions for 1,407,120 missense mutations associated with 111 protein structures from 163 inherited eye diseases. However, further progress in a building of the ocular proteome database is limited due to the computational complexity of the algorithm and a large volume of data.